This laboratory continues to examine the role of pteridine and folate coenzymes or substrates in biological systems. Naturally occurring pteridines or folates usually contain 2-amino and 4-hydroxy substituents, a reduced pyrazine ring, and often an alkyl substituent at the 5-position. During the course of a pteridine- or folate-mediated enzymatic reaction, oxidation of the reduced pyrazine ring can occur and in some cases the 5-substituent is lost or transferred. Our interests in the biochemistry of these compounds lie in three areas: (a) The efficient, quantitative isolation by affinity chromatography of a group of pteridine or folate requiring enzymes, which includes dihydropteridine reductase, phenylalanine hydroxylase, tyrosine hydroxylase, dihydrofolate reductase, and thymidylate synthetase, followed by their structural characterization using both conventional procedures and newly synthesized fluorescent and photolabile probes; (b) A study of the chemistry of the reduced and substituted pteridines using (1H) and (13C) NMR, and rapid reaction techniques (e.g., stop-flow spectrophotometry), with particular emphasis on the structural changes which induce lability into a 5-substituent, and (c) The preparation of high molecular weight pteridine or folate derivatives which may have chemotherapeutic utility.